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On-Site Confirmation and Monitoring

On-Site Confirmation and Monitoring. Cor Schoen. 29 th Nov, 2010 Q-detect. WP7. On-Site Confirmation and Monitoring. Main objective: To develop rapid, simple and reliable confirmatory and monitoring methods based on the detection of DNA. Overview of work for Task 7.

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On-Site Confirmation and Monitoring

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  1. On-Site Confirmation and Monitoring Cor Schoen 29th Nov, 2010 Q-detect

  2. WP7. On-Site Confirmation and Monitoring • Main objective: To develop rapid, simple and reliable confirmatory and monitoring methods based on the detection of DNA

  3. Overview of work for Task 7 Task 7.1 DNA extractions Task 7.2 Generation of protocols for isothermal, singleplex and multiplex amplification of targets Different isothermal amplification strategies will be tested for on-site confirmation: LAMP, NAIMA and PLP-RCA. Task 7.3 Generation of devices for multiplex detection on-site Task 7.4 Evaluation of promising on-site detection systems for monitoring This task may comprise the following subtasks: 1) evaluate selected robust DNA extraction procedure for each pathosystem 2) compare classical PCR to selected isothermal methods 3) select optimal single-plex and multiplex detection methods 4) establish one protocol with strong focus on simplicity Task 7.5 Implementation of procedure on user-friendly detection system

  4. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site White flies viruses from traps Bacterial pathogens from plant material Potato pathogens from tuber and leaves DNA/RNA Extraction Criteria: - Easy - Fast - Efficient - Cheap

  5. With respect to simplicity, speed and pricing, purification of nucleic acid (RNA and DNA) based on lateral flow devices would be ideal. Boonham et al 2008

  6. Boonham et al 2008

  7. Boonham et al 2008

  8. Conclusion extractions: • Qiagen DNeasy/RNeasy extraction kids have been successfully used for most of the described targets in WP7 • LFD based DNA/RNA extractions: • Easy to perform • Fast • Applicable for different substrates • (RNA and DNA viruses has to be tested) • Recovery has to be improved

  9. To validate LAMP, NAIMA and PLP-RCA, different TaqMan assays were developed as the golden standard for target detection:

  10. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site White flies viruses from traps Bacterial pathogens from plant material Potato pathogens from tuber and leaves • Monitoring Isothermal multiplex and simplex amplification LAMP (Optisense) NAIMA ? • Confirmation • Isothermal multiplex and simplex amplification • LAMP (Optisense) • PLP in combination with RCA (Qlinea)

  11. Extraction DNA / RNA React.Mix First line screening (monitoring via fast semi specific method) - + Χ Second line screening (confirmation via target specific detection methods) Specific target detection Real positive? Confirmation with another technology

  12. Monitoring (first line screening) • Isothermal multiplex and simplex amplification • Easy • Fast • Sensitive • Applicable for DNA and RNA  - PCR - PLP - RCA - NAIMA - LAMP   

  13. Isothermal amplification LAMP – Loop mediated Amplification - Amplification at a constant temperature (65oC) using a DNA polymerase with strand displacement reaction - Amplification and detection of the target can be completed in a single step. - High amplification efficiency, with DNA being amplified 109- 1010 times in 15- 60 minutes

  14. LAMP characteristics (summary) • There is no need for a step to denature double stranded into a single stranded form. • The whole amplification reaction takes place continuously under isothermal conditions. • Amplification can be done with RNA templates following the same procedure as with DNA templates, simply through the addition of reverse transcriptase. • The amplification efficiency is extremely high (10x higher than PCR). • LAMP is less prone to inhibitors • By designing 4 primers to recognize 6 distinct regions, the LAMP method is able to specifically amplify the target gene. • The total cost can be reduced, as LAMP does not require special reagents or sophisticated equipments. • The amplified products have a structure consisting of alternately inverted repeats of the target sequence on the same strand.

  15. Monitoring of LAMP products

  16. Quantitative analysis by real-time turbidimetry

  17. Multiplex LAMP product measurementwith micro-wells or micro-fluidics

  18. ABI microfluidic plate with prespotted primers: - 8 samples can be tested for 48 differenttargets. - Does not require liquid handling robotics - Provides easy standardization

  19. Detection of LAMP products LAMP product monitoring (summary) • (By-)products of LAMP (pyro phosphate) can be used for target detection and quantification • ‘Multiplex LAMP’ can be performed in microwells and different micro- fluidic devices

  20. Confirmation and Monitoring • DNA/RNA extractions on different substrates to be performed in the field and/or on-site White flies viruses from traps Bacterial pathogens from plant material Potato pathogens from tuber and leaves • Monitoring Isothermal multiplex and simplex amplification LAMP (Optisense) • Confirmation • Isothermal multiplex and simplex amplification • LAMP (Optisense) • PLP in combination with RCA (Qlinea)

  21. First line screening (monitoring via fast semi specific method) Second line screening (confirmation via target specific detection methods) Real positive? Confirmation with another technology

  22. Ligation based Univ-LAMP (LU-LAMP) for point mutation specific multiplexing Target ligation Biotin/Strep binding LAMP detection LAMP detection LAMP detection Capturing LAMP ampl/cutting LAMP ampl/cutting LAMP ampl/cutting Washing/ release

  23. Ligation based Univ-LAMP (LU-LAMP) for point mutation specific multiplexing Target ligation with universal LAMP primers Exonuclease cutting LAMP detection + LAMP LAMP amplicon cutting

  24. PLP based multiplex detection

  25. Mechanism for amplified single molecule detection

  26. Working mechanism for amplified single molecule detection

  27. RCA based quantitative multiplex detection Target recognition and circle creation Microfluidic analysis Signal amplification and specific labeling Thresholding Sample

  28. LAMP product confirmation • • Ligation based spec-LAMP (LS-LAMP) will probably support point mutation specific discrimination of targets in different array formats • • Ligation based Univ-LAMP (LU-LAMP) will be tested • PLP RCA based confirmation • • High level of specificity and multiplexing • Target recognition and amplification independent • Universal downstream processing after ligation • Ligation Fluorescently stained ‘blobs’ can easily be discriminated and identified in a micro-fluidic device • NAIMA confirmation • Improvement of NAIMA universal amplification primers • Hexaplex NAIMA amplification confirmed • Microarray platform detection experimented on hexaplex NAIMA

  29. Deliverables D 7.1 Three protocols for DNA extraction for selected set of targets in the different matrices (Month 12) D 7.2 Provide information to WP2 (partner 9) on the cost, time, skill and conditions required for the application of the detection method by inspectors. And preliminary values for parameters (or estimates) for uncertainties, sampling, quality (sensitivity/specificity) and protocols (Month 12). D 7.3 Two protocols for isothermal amplification of selected targets (Month 24) D 7.4 Three protocols for single-plex/multiplex analysis of selected targets (Month 30) D 7.5 Provide information to WP2 (partner 9) on the uncertainties, sampling, quality (sensitivity/specificity) and a full protocol required for the detection method developed (Month 30). D 7.6 Two Devices to be used on-site (Month 30) D 7.7 Two monitoring protocols (Month 36)

  30. Task 7.1 DNA extractions: (Month 12) • Three novel extraction approaches (devices, set-ups, reagents) will be developed. • Robustness of the three most promising extraction procedures will be determined. • Applicability of three different extraction procedures for the full-range of material encountered by end-user inspectors (fruit tree material – flowers, shoots, rootstocks, branches, fruit; potato leaf, wood shavings (for PWN), vector insects (e.g. whitefly and Monochamus beetles) and isolated nematodes (e.g. trapped PWN). will be tested). • Select and validate three on-site DNA extraction procedures to optimize assay sensitivity. Modification of three proven DNA extraction methods towards simplest possible manipulation for direct-in field application (e.g., one-step sample/DNA extraction in the field). This part has potential application for all project organisms. • The three best candidate methods will be evaluated in the different laboratories.

  31. Task 7.1 DNA extractions (continued): The following pathosystems will be addressed in this WP: PRI: Targets pests will be white flies (Bemisia tabaci) from traps ACW: Target pests will be bacterial fruit-tree pathogens (Erwinia amylovora – pome fruit; Xanthmonas arboricola pv. pruni – stone fruit). NIB: Target pests for testing DNA extraction will be the potato brown rot (Ralstonia solanacearum) and the potato ring rot (Clavibacter michiganensisssp. sepedonicus) on potato leaves and tubers. Fera:Targets will be PWN directly on trapped nematodes, within vector beetles and wood shaving material and potato leaves. UNIBO: Will participate on the development of the extraction protocols for all the bacterial species considered. In addition, UNIBO will also focus on Pseudomonas syringae pv actinidiae, an emerging pathogen that is jeopardizing kiwifruit production in south Europe. A new type of extraction and amplification of target will be performed. CIP:Focus on DNA/RNA extraction of PYVV form infected potato leaves. CAIQ:Focus on DNA/RNA extraction of Cotton leaf curl virus, transmitted by white flies and also on Citrus greening.

  32. Task 7.2 Generation of protocols for isothermal, singleplex and multiplex amplification of targets:(Month 24). • Two different isothermal amplification strategies will be tested for on-site confirmation: LAMP and NAIMA. • Optimized single-plex reaction procedures will be established for three key target pests (B. tabaci, Erwinia amylovora, Potato yellow vein virus) to enable assessing the performance of multiplex methods and to test limits of single-plex PCR methods with respect to speed and sensitivity.

  33. Task 7.2 Generation of protocols for isothermal, single-plex and multiplex amplification of targets:(Month 24). • Multiplex reaction procedures using diverse technologies will be developed. The padlock probes technology will be investigated for simultaneous on-site detection of viruses (e.g. TYLCV, CYSDV, PYVV, TICV, ToCV, begomoviruses and including cotton leaf curl virus) within white flies (Bemisia tabaci). The combination of multiplex NAIMA amplification with easy-to-use portable detection systems will be developed for simultaneous detection of DNA and RNA pathogens. The target candidates will be the bacteria Ralstonia solanacearum and Clavibacter michiganensis ssp. sepedonicus, and one viral pathogen to be determined (Potato spindle tuber viroid (PSTVd), the Potato Virus Y (PVY) or the Pepino Mosaic Virus (PepMV)). The main focus will be placed on the robustness of the amplification process under variable conditions. Enzymes, buffers and amplification conditions (temperature, hold times, reagents etc) will be tested. • PRI, Fera, ACW and NIB will collaborate in the testing and evaluation process.

  34. Task 7.3 Generation of devices for multiplex detection on-site(Month 24) • Qlinea will develop a prototype of a multiplex (10-20) single molecule detection device to be used for on-site application • Optisense will provide access to a portable isothermal amplification reader to WP6 partners for validation purposes. • PRI and Qlinea will collaborate in testing and evaluating the device developed by Qlinea for Bemisia transmitted virus detection. • Fera and Optisense will collaborate in testing and evaluating the devices developed by Optisense for PWN and PYVV detection

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